Feed and feed additive for marine species and crawfish and method for the production of same

ABSTRACT

The invention relates to a new feed, feed additive and method for production of feed for marine species. Trimethylamine oxide (TMAO) or deriviatives of same are formed to be powerful iron inhibitors when added to a conventional feed composition. 
     When used alone or in diluted dry or liquid form the amount needed to obtain an optional synergetic effect between iron and omega-3 fatty acids in the feed can be calculated according to a defined equation.

The present invention relates to a feed and a method for the productionof a feed for marine species, fish and crawfish, as well as a specialadditive which reduces the availability of the mineral iron in the feed.

The abundant access to marine raw materials in the form of fish meal,has resulted in that the (Norwegian) aquaculture industry to a greatextent has used such raw materials in feed blends. Additionally fishmeal has proved to be especially well suited as a source of proteins inthe feed. Analysis of commercial fish meal products show greatvariations in the iron content, normally from 140 to 300 mg total ironper kg meal. This is the main cause for the relatively high and varyingcontent of iron in commercial fish feed products, where there are normalvariations from 200 to 400 mg total iron per kg feed. Because iron inraw materials in general is less available, inter alia because of thecontent of phosphates, phytates and oxalates, than iron from animal rawmaterials, the use of marine raw materials has lead to a high content ofbioavailable iron in feed used by the aquaculture industry. However,this has been considered as an additional advantages of using such rawmaterials for the production of fish feed.

In our prior patent application nr. EPO NO 902274 it was shown that thelevel of iron in the existing feed for farmed salmon is far too high,and that this will reduce the growth and even increase the risk fordiseases.

To obtain an optimal synergy between iron and omega-3 fatty acids it wasshown that even the lowest level of 120 mg total iron per kg feed can betoo high, in spite of the fact that this level of iron is significantlylower than that which is generally considered as required. Cfr. WatanabeNutrient requirements of cold water fishes, No 16, National AcademyPress, Washington DC, 1981, where levels of iron from 250-400 mg iron/kgfeed is recommended.

In carrying out additional tests we have now found that there is presentapproximately three times as much iron in the liver in cultured salmonas in the liver of wild salmon. Excess iron is stored in the liverrather than being used for heamoglobine synthesis. This confirms thatthere exists a significant iron unbalance in present day fish feed.

Thus, it is the object of the invention to provide a feed and toestablish conditions for feeding which will lead to a reduction of theavailability of the mineral iron in the feed in comparison with astandard feed.

To improve the iron balance it is necessary to reduce the amount of ironin the feed. A reduction of the iron content of the feed toapproximately 100 mg / kg can be done by selecting and using commercialfish meal products having a lowest possible content of iron.Additionally there must be used mineral blends without iron addition.This very time consuming and economically costly method, appears todayas the only possibility to produce a commercial fish meal based feedwith an iron content of approximately 100 mg iron/kg dry feed.

Due to the fact that an additional lowering of the iron content isnecessary, this can only be obtained through the use of speciallyselected protein products. This will make the feed so expensive that thewhole farming business may be uneconomical. There may also occur a lackof suitable protein raw materials, at the same time as this will havenegative economic consequences for the traditional fish meal producers.

Special studies have been initiated to, if possible, explain why farmedsalmon which is fed traditionally has such a pronounced iron unbalanceand a poorer health status than wild salmon.

There may basically be three explanations of the iron unbalance.

1. The wild fish is regulating its uptake of iron in an up to nowunknown way.

2. There is too high content of iron in the feed.

3. The availability of the iron present in the feed is too high.

Our prior extensive investigations on farmed salmon seem to indicatethat it does not have a regulating mechanism for iron uptake comparablewith what is known from mammals. It is natural to assume that this willalso apply for wild salmon and other wild fish species.

Because raw marine fish and crawfish is a natural feed for wild salmonand fish meal makes up a significant part of commercial fish feed, wehave presumed that the main difference between the feed of wild salmoncompared with farmed salmon is not the total content of iron only butalso the availability of the iron present.

Chelators is a common denomination for all substances which are able toform complexes with iron, and thereby to reduce the availability of themineral. Damaging effects caused by too high iron doses can thereforpossibly be eliminated through the use of chelators or inhibitors foriron absorption and result in a greater part of the iron being excretedwithout being absorbed.

We have developed the hypothesis that the iron unbalance is createdbecause the iron in commercial fish meal is more readily available thanthe iron in raw fish. The reason for this is that a raw marine fish hasa high natural of the N-containing base trimethylamine-oxide (TMAO,(CH₃)₃ NO). TMAO is known for its potent precipitation of iron. BecauseTMAO is naturally available in all marine fishes and crawfish, it isalso a natural part of the feed used by the wild salmon.

Furthermore TMAO is detected in the intestines of fish, which is theplace where the uptake of iron is occurring. A number of analyses haveadditionally shown that the TMAO content in fish will increase withdecreasing sea temperatures With basis in our prior discoveries, cfr.the above mentioned patent application 902274, it is because of theselow sea that it of special importance for the salmon to reduce theuptake of iron. The reason is that in vitro studies have shown, that thered blood cells will agglutinate and form a clot when the blood containsmore iron than the transport mechanisms (serumtransferrin) can take careoff. This can lead to thrombosis in the fish. Thrombosis is, accordingto our experience, probably the cause of winter ulcers in the fish.Winter ulcers are surface scars on the side of the salmon. By low seatemperatures they occur frequently and have detrimental economicalconsequences, because the ulcers give a high mortality and theslaughtered fish is degraded to production fish.

Thus, while wild fish is consequently taking up low amounts of iron fromits natural feed, this is not the case for farmed salmon.

The reason for this is that TMAO in processed fish feed does not havethe ability to precipitate iron. This is the case because TMAO undergoeschanges by heating. Under production of fish meal, the fish is heatedover prolonged time. During the feed production the raw material isadditionally heated passing through an extruder or other processingapparatus followed by a final drying step of remove remaining water.

We have now surprisingly found that the addition of synthetic TMAO willin fact lower the iron absorption in farmed salmon. The salmon was fedwith the iron isotope ⁵⁹ Fe, with or without addition of synthetic TMAO,and the blood was analyzed after 24 hours. We found marked lowerradioactivity in those fishes which had received TMAO additive comparedwith those who did not receive TMAO additive. This illustrates that TMAOwill lower the iron absorbtion in farmed salmon.

The marked difference between iron availability in raw and processed(cooked) fish is documented within the fur breeding industry. In thisconnection we refer to A. Skrede: Meldinger fra Norges Landcrukshgskolevol. 62, nr. 4. Here it is documented that mink fed with raw fishacquired anemia (low levels of blood haemoglobin) which caused whiteunderfur, resulting in a product without economic value. Mink which werefed with boiled fish did however not develop anemia and had a normalfur.

The reason for this is that raw fish has a high content of active TMAO.TMAO caused anemia because free iron was transferred to undissolvableironoxihydroxide. These experiences were made for the first time withinthe fur industry 20-25 years ago and relates to lack of bioavailableiron in mink feed.

Our new knowledge pertaining to the absence an iron uptake regulatingmechanism in farmed salmon indicates that a feed for the fish farmingindustry will need a different approach: how can the amounts ofbioavailable iron be reduced to obtain an optimal uptake of iron.

Thus it is the main object of the present invention to provide a fishfeed where the amount of bio available iron can be controlled. Anotherobject is to allow for the continued use of marine fish meal as aprotein source for fish feed. Another object of the invention is toprovide a fish feed and a method for producing it, where the amount ofbiolavailable iron is controlled to obtain an optimal synergetic effectbetween iron and the n-3 fatty acids present.

A further object of the invention is to provide an additive which cancontrol the amount of bioavailable iron.

These and also other objects are obtained with the invention as definedin the accompanying patent claims and which is essentially characterizedby the addition to and the use of TMAO in fish feed.

The invention will now be described in more detail in connection withthe results obtained in experiments 1-3 and as illustrated in thedrawings, where FIG. 1 shows the decreasing bioavailable of iron withincreasing additions of TMAO,

FIGS. 2a-2d show respectively, the reduced uptake of iron in the blood,liver, spleen and heart of farmed salmon when TMAO is present and

FIG. 3 shows that TMAO shows a preference towards iron compared withother essential minerals such as zinc and manganese.

RESULTS Trial 1

Twentyfive salmon were divided into 5 groups, each of 5 fish. They wereall fed a solution of 200 μg ferrous iron and 5 different levels of TMAO: 0, 5, 10, 20 and 40 times the amount of iron. All fish were bloodsampled after 24 hours for determination of content of radioiron in theblood. We observed a highly significant and linear correlation betweeniron absorbed and TMAO added in the final solution (r =-0.998, df =4,p<0.001, Table 1 and FIG. 1).

                  TABLE 1                                                         ______________________________________                                        Effect of increasing amount of TMAO in the feed                               solution on iron absorption in Atlantic salmon.                                                    Uptake of iron                                                                             Absorption                                  Proportion                                                                              Number of  in the blood relative to                                 TMAO to Fe                                                                              fish       g ± SD    control                                     ______________________________________                                        Control   5          4.16 ± 1.04                                                                             100.0%                                       5:1      4          3.60 ± 1.04                                                                             86.5%                                       10:1      5          3.20 ± 0.60                                                                             76.9%                                       20:1      5          2.52 ± 0.71                                                                             60.5%                                       40:1      4          0.98 ± 0.78                                                                             23.6%                                       ______________________________________                                    

Trial 2

The time of blood sampling may be critical. Therefore, to providedocumentation of the total effects of TMAO on iron uptake, blood samplesat different time intervals after feeding may be needed. In addition, itis urgent to measure the level of radioiron in liver and spleen of fishfed different levels of TMAO. If more radioiron is found in the spleenof fish fed no TMAO than in those fed TMAO, cell membrane damage in thered blood cells may be suggested.

Thirty salmon were divided into 2 groups of 15 fish each. All fish inthe control group were administrated a feed solution of 200μg ferrousiron without any TMAO. The remaining fish were fed the same amount ofiron together with 8 mg TMAO (TMAO : Fe =40 : 1).

Blood, liver and spleen were sampled from each group at 3 different timeintervals after feeding : ##STR1##

We observed reduced uptake of iron in blood, liver and spleen at alltime intervals after feeding for fish fed TMAO (Table 2 and FIG. 2). Thereduced uptake of iron in the TMAO group compared to the control groupwas significant at all time intervals and organs except for : bloodafter 48 hours (p<0.1), spleen after 48 hours and spleen after 96 hours(P<0.1).

Iron overload may be connected to heart disease in farmed Atlanticsalmon. Therefore, the iron taken up by the heart after 96 hours wasmeasured (Table 2). Fish fed TMAO in the diet had significantly reducediron level in the heart compared to fish in the control group (p<0.005).

                  TABLE 2                                                         ______________________________________                                        Levels of iron in blood, liver, spleen and heart at                           different time intervals after feeding in Atlantic                            salmon fed an iron solution with and without TMAO.                                       Sampling time after feeding                                                         24 hours   48 hours 96 hours                                 Organs Treatment (μg ± SD)                                                                          (μg ± SD)                                                                        (μg ± SD)                          ______________________________________                                        Blood  Control   3.43 ± 1.56                                                                           6.58 ± 4.85                                                                         8.85 ± 5.06                           Blood  TMAO      0.81 ± 0.39                                                                           1.71 ± 1.74                                                                         2.93 ± 1.14                           Liver  Control   2.18 ± 0.97                                                                           2.87 ± 0.65                                                                         3.31 ± 1.11                           Liver  TMAO      0.42 ± 0.12                                                                           0.81 ± 0.66                                                                         0.84 ± 0.19                           Spleen Control   0.08 ± 0.05                                                                           0.11 ± 0.04                                                                         0.26 ± 0.15                           Spleen TMAO      0.02 ± 0.01                                                                           0.09 ± 0.07                                                                         0.12 ± 0.04                           Heart  Control   not sampled                                                                              not sampled                                                                            0.029 ± 0.012                         Heart  TMAO      not sampled                                                                              not sampled                                                                            0.008 ± 0.002                         ______________________________________                                    

The number of fish was 5 in each treatment, except for TMAO/24 hours andControl/48 hours where the number was 4.

Trial 3

In trial 1 and 2, the effect of TMAO on iron uptake without anyinfluence of other minerals was studied. It is, however, of majorimportance to know if TMAO also affects the uptake of other minerals.Thus, we studied the effects of TMAO on a mixed diet containingradioactive isotopes of 3 different minerals.

Isotopes :

i) ⁶⁵ Zn

ii) ⁵⁴ Mn

iii) ⁵⁹ Fe

The fishes were fed a solution of 200 g iron, 150 g zinc and 50 gmanganese each. Two treatments were used, control and TMAO. The amountof TMAO added was 40 times that of iron, 53 times that of zinc and 160times that of manganese. The time of blood sampling was 24 hours afterfeeding In addition to the mixed mineral solution, individual studieswere conducted for iron, zinc and manganese.

Fish fed TMAO and a mixed solution of all minerals had significantlyreduced level of radioiron in the blood compared to fish in the controlgroup (p<0.001). TMAO did not reduce the uptake of zinc and manganese(Table 3, FIG. 3)

In the experiment with one single mineral in solution, TMAO reduced theuptake as follows: iron =82% (p21 0.05), zinc=30% (not significant) andmanganese=69% (not significant).

                  TABLE 3                                                         ______________________________________                                        Effects of TMAO on a mixed feed solution of iron,                             zinc and manganese.                                                                                       Reduced                                                             Blood level                                                                             absorption rela-                                  Mineral Treatment (g)       tive to control                                                                         Statistics                              ______________________________________                                        Iron    Control   10.44 ± 3.18                                             Iron    TMAO       2.10 ± 1.10                                                                         80%       p <0.001                                Zinc    Control   10.44 ± 2.05                                             Zinc    TMAO      10.05 ± 2.54                                                                         4%        n.s.                                    Manganese                                                                             Control    0.12 ± 0.03                                             Manganese                                                                             TMAO       0.12 ± 0.09                                                                         0%        n.s.                                    ______________________________________                                    

Standard fish feed compositions contain basically protein and lipids andsome carbohydrates as main components. Additionally other componentssuch as binding agents, preservatives, vitamins and minerals are added.The ingredients are mixed together in the required proportions and themixing can be performed with or without the addition of water or liquidto produce a dry, soft or wet feed. As protein source there ispreferably used fish meal and as lipid there is used marine fats andpreferably fish oils or fish oil concentrates. The mineral mix usedshould be without iron additives. Even if it requires considerable skilland know-how to produce a high quality feed, the basic process is wellknown in the prior art and need not be described in detail here. Byvarying the amount of fish oil or fish oil concentrates used, the n-3content of the feed can be adjusted to be in the area from 2.0% to 8.5%while the amount of iron will vary between 100-400 mg/kg dry feeddepending on the selected raw materials. Also the pH of the feed shouldnot be lower than 5.5, and preferably should be adjusted between 6-7.

The required amount of TMAO can be calculated on the basis of the curveand equation presented in FIG. 1. From regression equation, it may beestimated the amount of TMAO needed to reduce the uptake of iron the todesired level.

The TMAO is preferably in the form of trimethylamine-oxide modifiedmolecules, or a derivative of same in dry or liquid form having the samefunction. TMAO can be added preferably in the form of a dry powder withvarious amounts of TMAO or mixed with a dry fish meal. Alternatively itcan be dissolved and diluted in water or liquid and added to the feedmixture.

Based on the average content of iron and n-3 given above, using fishmeal as protein source and fish oil as liquid source and a pH, of 6.0the required amount of TMAO will be 3.8 g to obtain a reduction of ironfrom 150 to 75 mg/kg.

The importance of reducing the iron availability through the use of TMAOin the fish feed or through its use as an additive only related tofarmed salmon but also applies for other farmed marine species, forfeeding small fry as well as for feeding grown fish.

We claim:
 1. In a feed for fish and crawfish which contains protein,lipids and carbohydrates in combination with one or more additionalcomponents mixed together to make up a dry, soft or wet feed with acontent of n-3 fatty acids of from 2.0-8.5% based on the dry feed, theimprovement consisting essentially of the feed and trimethylamine-oxide,said trimethylamine - oxide present in a controlled iron inhibitingeffective amount based on the equation y=0.536×1,388 wherein y istrimethylamine-oxide and x is iron content.
 2. A feed according to claim1, wherein the additional components are selected from the groupconsisting of fillers, adhesives, preservatives, vitamins and minerals.3. A method for the production of a fish meal or feed for fish andcrawfish, which comprises mixing together proteins, lipids,carbohydrates and one or more additional components, determining thefeed's content of available iron, and adding trimethylamine-oxide to themixture to reduce the availability of iron, so that there is obtained anoptimal controlled content of available iron in the fish meal or feedbased on the equation y=0.536×-1.388 wherein y is trimethylamine-oxideand x is iron.
 4. A method according to claim 3 wherein the additionalcomponents are selected from the group c